Direct demonstration of lipid phosphorylation in the lipid bilayer of the biomimetic bicontinuous cubic phase using the confined enzyme lipid A phosphoethanolamine transferase

Abstract

Retention of amphiphilic protein activity within the lipid bilayer membrane of the nanostructured biomimetic bicontinuous cubic phase is crucial for applications utilizing these hybrid protein-lipid self-assembly materials, such as in meso membrane protein crystallization and drug delivery. Previous work, mainly on soluble and membrane-associated enzymes, has shown that enzyme activity may be modified when immobilized, including membrane bound enzymes. The effect on activity may be even greater for amphiphilic enzymes with a large hydrophilic domain, such as the Neisserial enzyme lipid A phosphoethanolamine transferase (EptA). Encapsulation within the biomimetic but non-endogenous lipid bilayer membrane environment may modify the enzyme conformation, while confinement of the large hydrophilic domain with the nanoscale water channels of a continuous lipid bilayer structure may prevent full function of this enzyme. Herein we show that NmEptA remains active despite encapsulation within a nanostructured bicontinuous cubic phase. Full transfer of the phosphoethanolamine (PEA) group from a 1,2-dioleoyl-glycero-phosphoethanolamine (DOPE) doped lipid to monoolein (MO), which makes up the bicontinuous cubic phase, is shown. The reaction was found to be non-specific to the alkyl chain identity. The observed rate of enzyme activity is similar to other membrane bound enzymes, with complete transfer of the PEA group occurring in vitro, under the conditions studied, over a 24 hour timescale.

Inorganic nanoparticles modify the phase behavior and viscoelastic properties of non-lamellar lipid mesophases

“…The presence of additives modifies and controls the geometry of these lipid architectures. This is the case for fatty acids [15,16], photo-switchable molecules [17][18][19], and proteins [20] that can be even crystallized within the lyotropic phase [21,22]. Recently, Briscoe et al [23,24] investigated the effects of hydrophobic silica NPs on dioleoyl-phopshatidylethanolamine mesophases, highlighting a temperature and pressure-dependent lamellar to hexagonal phase transition.…”

Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1

“…Previous work with Nm EptA enzyme showed that the PEA transferase is promiscuous with regard to the alkyl chain identity of the donor substrate PE as the enzyme, when encapsulated within a nanostructured bicontinuous cubic phase, transfers the PEA moiety from PE with unsaturated acyl chains (1,2-dioleoyl-glycero-phosphoethanolamine, or DOPE; 18:1-PE) as efficiently as that with saturated acyl chains (1,2-dimystroyl-glycero-phosphoethanolamine, DMPE; 14:0-PE) to the receiver substrate ( 30 ). Here, we also find that the length of the acyl chain of PE has little, if any, effect on the enzyme activity of MCR-1 ( Fig.…”

“…Therefore, MCR-1 transferase activity is specific to the PE as the donor substrate. Furthermore, in the work with Nm EptA enzyme, it was found that Nm EptA could transfer the PEA moiety from PE to monoolein to form monoolein phosphoethanolamine (MOPE) ( 30 ). Considering that monoolein, which is a monoacylglycerol, is structurally distinct from lipid A by both acyl chain tail and head groups, Nm EptA may have no specificity on the PEA receiver substrate ( 30 ).…”

“…Furthermore, in the work with Nm EptA enzyme, it was found that Nm EptA could transfer the PEA moiety from PE to monoolein to form monoolein phosphoethanolamine (MOPE) ( 30 ). Considering that monoolein, which is a monoacylglycerol, is structurally distinct from lipid A by both acyl chain tail and head groups, Nm EptA may have no specificity on the PEA receiver substrate ( 30 ). Therefore, determining whether MCR-1 can transfer the PEA group from PE to lipidic substrates besides lipid A is worthy of further investigation.…”

Effect of Cholesterol on Biomimetic Membrane Curvature and Coronavirus Fusion Peptide Encapsulation

“…They have well-defined water channels that can encompass the protein’s hydrophilic domains. Their well-defined X-ray and neutron scattering patterns allow for detailed investigation of their nanostructure upon protein and peptide encapsulation, or buffer and temperature changes. ,− Dispersed cubic phase nanoparticles (cubosomes) can be used as drug delivery vehicles and have a significantly larger lipid bilayer surface area to volume ratio compared to vesicles. , Cubosomes can preferentially accumulate at infected sites and can provide an extended circulation time while still allowing clearance from the body after one to several days . The lipid composition and charge of the nanoparticles are of significant importance to selectively target bacterial cells with a more negatively charged cell membrane while leaving healthy mammalian cells unaffected .…”